Epithelial Dysregulation Research Articles

The role of cGAS in epithelial dysregulation in inflammatory bowel disease and gastrointestinal malignancies.

The gastrointestinal tract is lined by an epithelial monolayer responsible for selective permeability and absorption, as well as protection against harmful luminal contents. Recognition of foreign or aberrant DNA within these epithelial cells is, in part, regulated by pattern recognition receptors such as cyclic GMP-AMP synthase (cGAS). cGAS binds double-stranded DNA from exogenous and endogenous sources, resulting in the activation of stimulator of interferon genes (STING) and a type 1 interferon response. cGAS is also implicated in non-canonical pathways involving the suppression of DNA repair and the upregulation of autophagy via interactions with PARP1 and Beclin-1, respectively. The importance of cGAS activation in the development and progression of inflammatory bowel disease and gastrointestinal cancers has been and continues to be explored. This review delves into the intricacies of the complex role of cGAS in intestinal epithelial inflammation and gastrointestinal malignancies, as well as recent therapeutic advances targeting cGAS pathways.

IL-4Rα signaling promotes barrier-altering oncostatin M and IL-6 production in aspirin-exacerbated respiratory disease

Aspirin-exacerbated respiratory disease (AERD) is a severe disease involving dysregulated type 2 inflammation. However, the role other inflammatory pathways play in AERD is poorly understood. We sought to broadly define the inflammatory milieu of the upper respiratory tract in AERD and to determine the effects of IL-4Rα inhibition on mediators of nasal inflammation. Twenty-two AERD patients treated with dupilumab for 3 months were followed over 3 visits and compared to 10 healthy controls. Nasal fluid was assessed for 45 cytokines and chemokines using Olink Target 48. Blood neutrophils and cultured human mast cells, monocytes/macrophages, and nasal fibroblasts were assessed for response to IL-4/13 stimulation invitro. Of the nasal fluid cytokines measured, nearly one third were higher in AERD patients compared to healthy controls, including IL-6 and the IL-6 family-related cytokine oncostatin M (OSM), both of which correlated with nasal albumin levels, a marker of epithelial barrier dysregulation. Dupilumab significantly decreased many nasal mediators, including OSM and IL-6. IL-4 stimulation induced OSM production from mast cells and macrophages but not from neutrophils, and OSM and IL-13 stimulation induced IL-6 production from nasal fibroblasts. In addition to type 2 inflammation, innate and IL-6-related cytokines are also elevated in the respiratory tract in AERD. Both OSM and IL-6 are locally produced in nasal polyps and likely promote pathology by negatively affecting epithelial barrier function. IL-4Rα blockade, although seemingly directed at type 2 inflammation, also decreases mediators of innate inflammation and epithelial dysregulation, which may contribute to dupilumab's therapeutic efficacy in AERD.

Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy.

Chronic rhinosinusitis with nasal polyp (CRSwNP) is a typical type 2 inflammation involving interleukin (IL)-4 and IL-13. Dupilumab is a fully human monoclonal antibody targeting IL-4 receptor α subunit, thereby blocking signaling by both cytokines. Our hypothesis was that IL-4 and IL-13, by inducing a severe epithelial dysregulation, are involved in CRSwNP pathogenesis. This study aimed to evaluate the in vitro direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions. Nasal polyps and control mucosa from 28 patients, as well as human nasal epithelial cells (HNEC) from 35 patients with CRSwNP were used. Three major epithelial functions were investigated: the epithelial barrier function (characterized by transepithelial electrical resistance measurements and tight junction protein expression), the ciliary motion (characterized by the ciliary beating efficiency index), and wound healing (characterized by the wound repair rate) under various stimulations (IL-4, IL-13, and dupilumab). The main outcome was a significant change in epithelial functions following exposure to IL-4, IL-13, and dupilumab for 48h in the basal media. IL-4 (1, 10, and 100ng/mL) but not IL-13 induced a significant decrease in occludin and zonula-occludens protein expression, ciliary beating efficiency, and wound repair rate in HNEC. Dupilumab (0.04mg/mL) had no effect on HNEC and specifically restored all epithelial functions altered when cells were exposed to a 48-h IL-4 stimulation. Dupilumab, in vitro, restored epithelial integrity by counteracting the effect of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased wound repair rate).

Expression profiling identifies key genes and biological functions associated with eosinophilic esophagitis in human patients.

Eosinophilic Esophagitis (EoE) is a chronic allergic disease characterized by progressive inflammation of the esophageal mucosa. This chronic inflammatory disorder affects up to 50 per 100,000 individuals in the United States and Europe yet is limited in treatment options. While the transcriptome of EoE has been reported, few studies have examined the genetics among a cohort including both adult and pediatric EoE populations. To identify potentially overlooked biomarkers in EoE esophageal biopsies that may be promising targets for diagnostic and therapeutic development. We used microarray analysis to interrogate gene expression using esophageal biopsies from EoE and Control subjects with a wide age distribution. Analysis of differential gene expression (DEGs) and prediction of impaired pathways was compared using conventional transcriptome analysis (TAC) and artificial intelligence-based (ADVAITA) programs. Principal Components Analysis revealed samples cluster by disease status (EoE and Control) irrespective of clinical features like sex, age, and disease severity. Global transcriptomic analysis revealed differential expression of several genes previously reported in EoE (CCL26, CPA3, POSTN, CTSC, ANO1, CRISP3, SPINK7). In addition, we identified differential expression of several genes from the MUC and SPRR families, which have been limited in previous reports. Our findings suggest that there is epithelial dysregulation demonstrated by DEGs that may contribute to impaired barrier integrity and loss of epidermal cell differentiation in EoE patients. These findings present two new gene families, SPRR and MUC, that are differentially expressed in both adult and pediatric EoE patients, which presents an opportunity for a future therapeutic target that would be useful in a large demographic of patients.

Modulation of Neuroimmune and Epithelial Dysregulation in Patients With Moderate to Severe Prurigo Nodularis Treated With Nemolizumab

Prurigo nodularis (PN) is a debilitating skin disease characterized by intense pruritus and hyperkeratotic skin nodules. Nemolizumab, a monoclonal antibody targeting interleukin 31 receptor α, is a promising novel therapy for the treatment of moderate to severe PN. The biological mechanisms by which nemolizumab promotes improvement of itch and skin lesions in PN are unknown. To characterize changes in plasma protein biomarkers associated with clinical response to nemolizumab in patients with PN. This multicenter cohort study included patients recruited from Austria, France, Germany, Poland, and the US from a phase 2 clinical trial. Adults diagnosed with moderate to severe PN with severe pruritus for at least 6 months were included in the original trial. Patients in the nemolizumab group were included in the present study if they achieved at least a 4-point decrease in the Peak Pruritus Numerical Rating Scale (PP-NRS) from baseline to week 12 during nemolizumab treatment. Placebo controls did not experience a 4-point decrease in PP-NRS. Mass spectrometry with tandem mass tags to enrich skin-specific protein detection was used to characterize changes in plasma protein expression in nemolizumab and placebo groups. Data were collected from November 2, 2017, to September 26, 2018, and analyzed from December 6, 2019, to April 8, 2022. As part of the clinical trial, patients were treated with 3 doses of nemolizumab or placebo at 0, 4, and 8 weeks. Changes in plasma and epidermal protein expression in nemolizumab-treated patients compared with the placebo group at 0, 4, and 12 weeks. Among the 38 patients included in the analysis (22 women and 16 men; mean [SD] age, 55.8 [15.8] years), enrichment analysis of canonical pathways, biological functions, and upstream regulators showed downregulation of terms involving inflammation (IL-6, acute-phase response, signal transducer and activator of transcription 3, and interferon γ), neural processes (synaptogenesis signaling and neuritogenesis), tissue remodeling and fibrosis (transforming growth factor β1 and endothelin-1), and epidermal differentiation (epithelial mesenchymal transition) in the plasma of nemolizumab group. In this cohort study, differences between nemolizumab and placebo groups included modulation of inflammatory signaling, neural development, and epithelial differentiation, suggesting a promising potential approach for clinical management of PN.

Tetramethylpyrazine Alleviates Tight Junction Disruption of Bronchial Mucosal Epithelial Cells Caused by Interleukin-17 via Inhibiting Nuclear Factor-κB-p65/Tumor Necrosis Factor-α Signaling Pathway.

Bronchial mucosal epithelial dysregulation and barrier disruption are involved in the initiation and development of acute lung injury (ALI). Some inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-17 (IL-17) contribute to the pathological changes of ALI. However, the roles and relationship between TNF-α and IL-17 during the disruption of bronchial epithelial tight junction remain unclear. Tetramethylpyrazine (TMP) is confirmed to have beneficial functions in hemostasis, inflammation, and cell growth. Here, we demonstrated the protective effects of TMP on bronchial mucosal epithelial injury induced by IL-17. We showed that IL-17 stimulation in vitro markedly reduced occludin and zonula occludens-1 (ZO-1) expression in bronchial mucosal epithelial cells via the nuclear factor-κB-p65/TNF-α signaling pathway, including NF-κB-p65-triggered TNF-α gene transcription and expression. TMP obviously rescued IL-17-induced occludin and ZO-1 downregulation. Mechanically, TMP substantially suppressed NF-κB-p65 activation and NF-κB-p65-induced TNF-α production in bronchial mucosal epithelial cells caused by IL-17. Taken together, this study indicates that TMP has a protective effect on bronchial mucosal epithelial cell injury due to IL-17 induction by inhibiting the NF-κB-p65/TNF-α signaling pathway.

Cluster Analysis of Inflammatory Biomarker Expression in the International Severe Asthma Registry

Allergy, eosinophilic inflammation, and epithelial dysregulation are implicated in severe asthma pathogenesis. We characterized biomarker expression in adults with severe asthma. Within the International Severe Asthma Registry (ISAR), we analyzed data from 10 countries in North America, Europe, and Asia, with prespecified thresholds for biomarker positivity (serum IgE ≥ 75 kU/L, blood eosinophils ≥ 300 cells/μL, and FeNO ≥ 25 ppb), and with hierarchical cluster analysis using biomarkers as continuous variables. Of 1,175 patients; 64% were female, age (mean ± SD) 53 ± 15 years, body mass index (BMI) 30 ± 8, postbronchodilator forced expiratory volume in 1 second (FEV1) predicted 72% ± 20%. By prespecified thresholds, 59% were IgE positive, 57% eosinophil positive, and 58% FeNO positive. There was substantial inflammatory biomarker overlap; 59% were positive for either 2 or 3 biomarkers. Five distinct clusters were identified: cluster 1 (61%, low-to-medium biomarkers) comprised highly symptomatic, older females with elevated BMI and frequent exacerbations; cluster 2 (18%, elevated eosinophils and FeNO) older females with lower BMI and frequent exacerbations; cluster 3 (14%, extremely high FeNO) older, highly symptomatic, lower BMI, and preserved lung function; cluster 4 (6%, extremely high IgE) younger, long duration of asthma, elevated BMI, and poor lung function; cluster 5 (1.2%, extremely high eosinophils) younger males with low BMI, poor lung function, and high burden of sinonasal disease and polyposis. There is significant overlap of biomarker positivity in severe asthma. Distinct clusters according to biomarker expression exhibit unique clinical characteristics, suggesting the occurrence of discrete patterns of underlying inflammatory pathway activation and providing pathogenic insights relevant to the era of monoclonal biologics.

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using invitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrinα6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Epithelial and Immune Phenotyping of Distal Airway Cells in Lung Allograft Dysfunction

Airway epithelial injury is thought to be the final common pathway leading to airway fibrosis in chronic lung allograft dysfunction (CLAD). While cumulative post-transplant inflammatory insults represent CLAD risk factors, we lack adequate methods to measure epithelial changes during such events. We hypothesize that early detection of allograft epithelial dysregulation and injury will reveal relevant pathways in CLAD pathogenesis and predict subsequent CLAD. We aim to use transbronchial brushings (TBBr) to sample distal airway epithelial cells and assess their characteristics in the context of allograft dysfunction. In this study, TBBr are being collected from patients undergoing routine surveillance bronchoscopies at 3, 6, 9, and 12 months post-transplant, as well as patients with acute lung allograft dysfunction (ALAD), defined as a >=10% drop in FEV1 compared to previous baseline (Figure 1A). Multi-colour flow cytometry panels were developed to identify populations of epithelial and immune cells. Cell cytospins were assessed with immunofluorescence (Figure 1B-C). In this preliminary interim analysis of TBBr from 14 patients (6 stable and 8 with ALAD), we were able to identify important epithelial and mucosal immune cell subsets. In patients with ALAD, there was a trend towards decreased club cells, which act as epithelial cell progenitors and have anti-inflammatory properties (Figure 1D-E). Additionally, there was a trend towards increased epithelial cell expression of CD54 (ICAM), an activation marker (Figure 1E). In contrast, intraepithelial T and B lymphocytes were not found to be different in these patient groups. This preliminary study shows that TBBr can successfully isolate airway epithelial and mucosal immune cells for identification of specific cell populations. Follow-up studies from this project will include recruitment of additional subjects, longitudinal sample analyses, and association of cellular changes with later clinical outcomes.

Epithelial barrier repair and prevention of allergy.

Allergic diseases have in common a dysfunctional epithelial barrier, which allows the penetration of allergens and microbes, leading to the release of type 2 cytokines that drive allergic inflammation. The accessibility of skin, compared with lung or gastrointestinal tissue, has facilitated detailed investigations into mechanisms underlying epithelial barrier dysfunction in atopic dermatitis (AD). This Review describes the formation of the skin barrier and analyzes the link between altered skin barrier formation and the pathogenesis of AD. The keratinocyte differentiation process is under tight regulation. During epidermal differentiation, keratinocytes sequentially switch gene expression programs, resulting in terminal differentiation and the formation of a mature stratum corneum, which is essential for the skin to prevent allergen or microbial invasion. Abnormalities in keratinocyte differentiation in AD skin result in hyperproliferation of the basal layer of epidermis, inhibition of markers of terminal differentiation, and barrier lipid abnormalities, compromising skin barrier and antimicrobial function. There is also compelling evidence for epithelial dysregulation in asthma, food allergy, eosinophilic esophagitis, and allergic rhinosinusitis. This Review examines current epithelial barrier repair strategies as an approach for allergy prevention or intervention.

The Development of Steady-State Activation Hubs between Adult LTi ILC3s and Primed Macrophages in Small Intestine.

Group 3 innate lymphoid cells (ILC3s) are important for intestinal health, particularly in controlling inflammation in response to epithelial dysregulation, but their role during homeostasis remains less well understood. We generated IL-22 reporter mice to assess production of this key cytokine by ILC3s in the small intestine during development and under basal conditions. Although IL-22 is produced by a variety of lymphocyte populations, constitutively high IL-22 expression was limited to lymphoid-tissue inducer (LTi) cells residing in lymph node-like structures in the gut called solitary intestinal lymphoid tissues (SILT). Constitutive IL-22 expression was dependent on the microbiota and MyD88 signaling, appeared upon weaning, and was present across the spectrum of SILT, including in cryptopatches. Activated SILT LTi cells colocalized with a rare subpopulation of activated macrophages constitutively positive for IL-12/23 p40 and capable of activating neonatal LTi cells in response to TLR stimulus. Thus, weaning leads to the organization of innate immune activation hubs at SILT that mature and are continuously sustained by signals from the microbiota. This functional and anatomic organization constitutes a significant portion of the steady-state IL-23/IL-22 axis.

Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

Endogenous circadian clocks regulate 24-h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse-chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro, caspase activity was assayed in Per1/2-specific small interfering RNA-transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.-Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

Characterisation of the lymphoid stress surveillance response in human intestine

BackgroundThe human intestine harbours an immense repertoire of T cells, many having proved difficult to isolate and characterise. The lymphoid stress surveillance response (LSSR), whereby unconventional T cells respond to epithelial dysregulation in synchrony with innate myeloid cells, was first described in mice. We investigated the potential for LSSR in human intestine, and whether this response could contribute to gut inflammation. MethodsWe obtained colonic biopsy samples at endoscopy from healthy donors. We applied a novel explant culture system (from one described in human skin) that permits outgrowth of large numbers of highly viable T cells, to complete flow-cytometric phenotyping and functional studies. To validate our method we compared T cell isolation methods. FindingsValidation of our method demonstrated that cell yield was twice as high as with conventionally isolated cells. γδ T cells comprised a substantial, underappreciated proportion of total intestinal lymphocytes (mean 9·5%, SD 9). Gut γδ T cells expressed a tissue-resident memory phenotype, including the activating receptor NKG2D. As previously demonstrated by us in human skin, specific cytokines licensed such cells to make purely innate-like cytotoxic responses, both to recombinant NKG2D ligands and to colonic tumour cell lines. Responses to colonic tumour cell lines were abrogated by antibodies that block NKG2D, demonstrating common functional potentials at distinct epithelial surfaces. InterpretationThe study shows that the human intestine harbours a large number of tissue-resident γδ T cells that can make rapid, innate-like responses to epithelial cell dysregulation, akin to those shown in our laboratory both in vivo for their mouse counterparts, and in skin. We can now investigate the contribution of LSSR to inflammatory gastrointestinal diseases characterised by tissue dysregulation and the upregulation of stress ligands. FundingFrancis-Crick Institute - National Institute for Health Research Biomedical Research Centre (Clinical Research Training Fellowship), Wellcome Trust.

Role of LRIG1-dependent EGFR signalling on pathway inhibition in airway homoeostasis and lung cancer development

BackgroundLRIG1 is a negative regulator of epidermal growth factor receptor (EGFR) signalling, and regulates stem-cell compartments in skin and gut epithelial cells. Interestingly, LRIG1 is lost in samples from patients with preinvasive squamous cell lung cancer. Basal cells are the upper airway stem cells and the putative origin of this cancer. We aimed to delineate LRIG1 expression within the airway, its effect on the stem-cell compartment, and whether its loss causes preinvasive disease. MethodsLrig1 (EGFP-IRES-CreERT2) mice were used to determine LRIG1 expression in the airways with immunofluorescence and flow cytometry. Basal stem-cell expression of LRIG1 and correlation with cell proliferation were examined. LRIG1+ cells were isolated with flow cytometry, and colony-formation and spheroid-formation assays were used to compare LRIG1+ and LRIG1– basal cells. To lineage trace LRIG+ cells, Lrig1 mice were crossed with reporters and were transgene activated. In-vivo and ex-vivo studies were performed. Data are given as mean (SE). FindingsLRIG1 expression was detected throughout the airway epithelium and within 66·3% (3·01) of basal cells. This LRIG1+ subpopulation of basal cells showed increased proliferation compared with LRIG1– cells (12·52% [1·82] vs 6·27 [2·13], p=0·0156). When plated in colony-formation assays, flow-sorted LRIG1+ expressing basal cells showed increased colony-forming efficacy and also had a greater spheroid-forming capacity (spheroid-forming capacity 3·3% [0·7] vs 0·56 [0·26], p=0·031). Ex-vivo lineage tracing assessed with spheroid assays indicated the development of clonal patches derived from LRIG+ basal cells. Formation of in-vivo clonal patches also occurred. InterpretationOur initial data indicate that LRIG1 has a role in airway stem-cell progenitor regulation and that it marks a more proliferative basal cell population. These data highlight the need to further investigate how the loss of LRIG1 leads to epithelial dysregulation and development of malignancy in patients. FundingThe Wellcome Trust, Cancer Research UK.

Drivers of chronic rhinosinusitis: Inflammation versus infection

Studies of the underlying cause or causes of chronic rhinosinusitis (CRS) over the past 20 or more years have expanded from a focus on systemic immune and allergic mechanisms to an intense search for the underlying drivers of mucosal inflammation. These drivers involve mucosal inflammatory pathways that become activated by allergens, microbial stimuli, or poorly understood exogenous or endogenous stimuli. The holy grail in the study of CRS is to identify specific drivers of mucosal inflammation and translate these into more effective treatment for CRS. Certain deficiencies in local innate immunity have been described in patients with CRS that predispose to increased sinus mucosal bacterial colonization/infection, including deficient local production of antimicrobial lactoferrin and deficient functioning of the bitter taste receptor TAS2R38. Conversely, certain innate factors, namely IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), are elaborated by sinus epithelial cells in response to microbial stimulation or airway injury and promote local TH2 inflammation. The precise physiologic role of these factors in innate or adaptive immunity is unclear, although IL-33 might function as an alarmin triggered by damage-associated molecular patterns. The cytokines IL-25 and TSLP, similarly promote proinflammatory tissue responses. Another feature of epithelial dysregulation in patients with CRS is overproduction of eosinophil-promoting C-C chemokines by sinus epithelium, perhaps driven in part through innate stimuli, as well as TH2 cytokines, such as IL-13. Strategies to reduce the microbial stimulation of maladaptive TH2 inflammation or to suppress the local elaboration of TH2-promoting epithelial factors, such as IL-33, have potential therapeutic benefit in patients with CRS, although the extent to which this is realized in patient care remains limited at present. This rostrum will summarize my views on the major microbial drivers of mucosal inflammation and dysregulation of innate TH2-promoting factors in patients with CRS based on recent experimental data.

Epithelial decision makers: in search of the 'epimmunome'

Frequent microbial and nonmicrobial challenges to epithelial cells trigger discrete pathways, promoting molecular changes such as the secretion of specific cytokines and chemokines and alterations to molecules displayed at the epithelial cell surface. In combination, these molecules impose key decisions on innate and adaptive immune cells. Depending on context, those decisions can be as diverse as those imposed by professional antigen-presenting cells, benefiting the host by balancing immune competence with the avoidance of immunopathology. Nonetheless, this potency of epithelial cells is also consistent with the causal contribution of epithelial dysregulation to myriad inflammatory diseases. This pathogenic axis provides an attractive target for tissue-specific clinical manipulation. In this context, a research goal should be to identify all molecules used by epithelial cells to instruct immune cells. We term this the 'epimmunome'.

Role for epithelial dysregulation in early-onset colitis-associated colon cancer in Gi2-alpha−/− mice

Inflammatory bowel disease (IBD) is a risk factor for developing colorectal cancer but the mechanisms are poorly characterized. Mice lacking the G-protein alpha subunit Gi2-alpha spontaneously develop colitis and colon cancer with high penetrance. Compared to canonical Wnt/APC signaling-based animal models of colon cancer, the tumors in Gi2-alpha-/- mice more closely recapitulate the features of IBD-associated cancers seen in humans. They are predominantly right-sided, multifocal, mucinous, and arise from areas of flat dysplasia. In evaluating the potential contribution of epithelial Gi2-alpha signaling to this phenotype, we found that Gi2-alpha-/- colonic epithelium is hyperproliferative even before the onset of colitis, and resistant to the induction of apoptosis. We generated colon cancer cell lines overexpressing dominant-negative Gi2-alpha. Like other cells lacking Gi2-alpha, these cells release less arachidonic acid, an important antiinflammatory and epithelial growth regulator. They are also hyperproliferative and resistant to camptothecin-induced apoptosis and caspase-3 activation. The colitis-associated cancers in Gi2-alpha-/- mice appear very similar to those seen in human IBD patients, and Gi2-alpha is a direct negative regulator of colonic epithelial cell growth.

Hydrogen sulfide induces serum-independent cell cycle entry in nontransformed rat intestinal epithelial cells.

Hydrogen sulfide (H2S), produced by commensal sulfate-reducing bacteria, is an environmental insult that potentially contributes to chronic intestinal epithelial disorders. We tested the hypothesis that exposure of nontransformed intestinal epithelial cells (IEC-18) to the reducing agent sodium hydrogen sulfide (NaHS) activates molecular pathways that underlie epithelial hyperplasia, a phenotype common to both ulcerative colitis (UC) and colorectal cancer. Exposure of IEC-18 cells to NaHS rapidly increased the NADPH/NADP ratio, reduced the intracellular redox environment, and inhibited mitochondrial respiratory activity. The addition of 0.2-5 mM NaHS for 4 h increased the IEC-18 proliferative cell fraction (P<0.05), as evidenced by analysis of the cell cycle and proliferating cell nuclear antigen expression, while apoptosis occurred only at the highest concentration of NaHS. Thirty minutes of NaHS exposure increased (P<0.05) c-Jun mRNA concentrations, consistent with the observed activation of mitogen activated protein kinases (MAPK). Microarray analysis confirmed an increase (P<0.05) in MAPK-mediated proliferative activity, likely reflecting the reduced redox environment of NaHS-treated cells. These data identify functional pathways by which H2S may initiate epithelial dysregulation and thereby contribute to UC or colorectal cancer. Thus, it becomes crucial to understand how genetic background may affect epithelial responsiveness to this bacterial-derived environmental insult.

Trichuris muris: Host Intestinal Epithelial Cell Hyperproliferation during Chronic Infection Is Regulated by Interferon-γ

Artis, D., Potten, C. S., Else, K. J., Finkelman, F. D., and Grencis, R. K. 1999. Trichuris muris: Host intestinal epithelial cell hyperproliferation during chronic infection is regulated by interferon-γ. Experimental Parasitology92, 144–153. Chronic infection with the intestinal nematode Trichuris muris is associated with an inappropriate type 1 cytokine response (production of predominantly IFN-γ), whereas resistance to infection requires the induction of a protective type 2 response with the production of interleukin (IL)-4, IL-5, IL-9, and IL-13. T. muris inhabits an intracellular niche within murine intestinal epithelial cells of the caecum and in common with other intestinal helminth infections is associated with gross morphological changes in gut architecture. The purpose of this study was to characterise cytokine production during chronic infection in AKR and severe-combined-immunodeficient (SCID) mice and investigate what effect the anti-parasite response had on epithelial cell proliferation and so regulation of intestinal pathology. Pulse labeling with tritiated thymidine is employed to generate a sensitive cell position-linked proliferation index of the intestinal epithelium at various times postinfection. Infection in AKR mice is characterized by a marked elevation in antigen specific IFN-γ production from restimulated mesenteric lymph node cells and a significant increase in proliferation of pluripotent epithelial stem cells and transit cells within the crypts. Similarly, elevated IFN-γ production was observed in the mesenteric lymph nodes and intestinal mucosa of infected SCID mice, with epithelial cell hyperproliferation and the development of crypt hyperplasia in the caecum. Critically, in vivo depletion of IFN-γ during infection in SCID mice resulted in no significant increase in epithelial cell proliferation and effectively precluded the development of crypt hyperplasia without altering infection outcome. Taken together, the data provides the first detailed cell position linked analysis of epithelial dysregulation during chronic T. muris infection and identifies a critical role for IFN-γ, either directly or indirectly, in regulation of epithelial cell proliferation during the chronic intestinal inflammation associated with infection.